Systems and methods for amplifying nucleic acids

ABSTRACT

An apparatus for performing a thermocyclic process, such as amplifying DNA, includes a microfluidic chip with a channel formed therein and one or more thermal distribution elements disposed over portions of the chip. Each thermal distribution element is configured to distribute thermal energy from an external thermal energy source substantially uniformly over the portion of the chip covered by the thermal distribution element. The portion of the chip covered by the thermal distribution element thereby comprises a discrete temperature zone. Other temperature zones can be defined by other thermal distribution elements or by portions of the chip not covered by a thermal distribution element. The channel is configured so that a fluid flowing through the channel would enter and exit the different temperature zones a plurality of times, thereby alternately exposing the fluid to the temperature of each zone for a period of time required for the fluid to traverse the zone.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application a divisional of U.S. patent application Ser. No.12/144,328, filed on Jun. 23, 2008, which is incorporated by referencein its entirety.

BACKGROUND Field of the Invention

The present invention relates to systems and methods for amplifyingnucleic acids. In some embodiments, the present invention relates tosystems and methods for performing a real-time polymerase chain reaction(PCR) in continuous-flow microfluidic systems and to methods formonitoring real-time PCR in such systems.

Discussion of the Background

The detection of nucleic acids is central to medicine, forensic science,industrial processing, crop and animal breeding, and many other fields.The ability to detect disease conditions (e.g., cancer), infectiousorganisms (e.g., HIV), genetic lineage, genetic markers, and the like,may facilitate disease diagnosis and prognosis, marker assistedselection, correct identification of crime scene features, the abilityto propagate industrial organisms and many other techniques.Determination of the integrity of a nucleic acid of interest can berelevant to the pathology of an infection or cancer.

One of the most powerful and basic technologies to detect smallquantities of nucleic acids is to replicate some or all of a nucleicacid sequence many times, and then analyze the amplification products.PCR is perhaps the most well-known of a number of differentamplification techniques.

PCR is a powerful technique for amplifying short sections of DNA. WithPCR, one can quickly produce millions of copies of DNA starting from asingle template DNA molecule. PCR includes a three phase temperaturecycle of denaturation of DNA into single strands, annealing of primersto the denatured strands, and extension of the primers by a thermostableDNA polymerase enzyme. This cycle is repeated so that there are enoughcopies to be detected and analyzed.

In principle, each cycle of PCR could double the number of copies. Inpractice, the multiplication achieved after each cycle is always lessthan 2. Furthermore, as PCR cycling continues, the buildup of amplifiedDNA products eventually ceases as the concentrations of requiredreactants diminish.

For general details concerning PCR, see Sambrook and Russell, MolecularCloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (supplemented through 2005), and PCR Protocols A Guide toMethods and Applications, M. A. Innis et al., eds., Academic Press Inc.San Diego, Calif. (1990).

Real-time PCR refers to a growing set of techniques in which onemeasures the buildup of amplified DNA products as the reactionprogresses, typically once per PCR cycle. Monitoring the accumulation ofproducts over time allows one to determine the efficiency of thereaction, as well as to estimate the initial concentration of DNAtemplate molecules. For general details concerning real-time PCR seeReal-Time PCR: An Essential Guide, K. Edwards et al., eds., HorizonBioscience, Norwich, U.K. (2004).

Several different real-time detection chemistries now exist to indicatethe presence of amplified DNA. Most of these depend upon fluorescenceindicators that change properties as a result of the PCR process. Amongthese detection chemistries are DNA binding dyes (such as SYBR® Green)that increase fluorescence efficiency upon binding to double strandedDNA. Other real-time detection chemistries utilize Foerster resonanceenergy transfer (FRET), a phenomenon by which the fluorescenceefficiency of a dye is strongly dependent on its proximity to anotherlight absorbing moiety or quencher. These dyes and quenchers aretypically attached to a DNA sequence-specific probe or primer. Among theFRET-based detection chemistries are hydrolysis probes and conformationprobes. Hydrolysis probes (such as the TaqMan® probe) use the polymeraseenzyme to cleave a reporter dye molecule from a quencher dye moleculeattached to an oligonucleotide probe. Conformation probes (such asmolecular beacons) utilize a dye attached to an oligonucleotide, whosefluorescence emission changes upon the conformational change of theoligonucleotide hybridizing to the target DNA.

A number of commercial instruments exist that perform real-time PCR.Examples of available instruments include the Applied Biosystems PRISM7500, the Bio-Rad iCylcer, and the Roche Diagnostics LightCycler 2.0.The sample containers for these instruments are closed tubes whichtypically require at least a 10 μl volume of sample solution. If thelowest concentrations of template DNA detectable by a particular assaywere on the order of one molecule per microliter, the detection limitfor available instruments would be on the order of tens of targets persample tube. Therefore, in order to achieve single molecule sensitivity,it is desirable to test smaller sample volumes, in the range of 1-1000nl.

More recently, a number of high throughput approaches to performing PCRand other amplification reactions have been developed, e.g., involvingamplification reactions in microfluidic devices, as well as methods fordetecting and analyzing amplified nucleic acids in or on the devices.Thermal cycling of the sample for amplification is usually accomplishedin one of two methods. In the first method, the sample solution isloaded into the device and the temperature is cycled in time, much likea conventional PCR instrument. In the second method, the sample solutionis pumped continuously through spatially varying temperature zones.

For example, Lagally et al. (Anal Chem 73:565-570 (2001)) demonstratedamplification and detection of single template DNA in a 280 nl PCRchamber. Detection of products was made post-PCR using capillaryelectrophoresis. On the other hand, Kopp et al. (Science 280:1046-1048(1998)) demonstrated continuous-flow PCR using a glass substrate with aserpentine channel passing over three constant temperature zones at 95°C. (denature), 72° C. (extension), and 60° C. (annealing). The 72° C.zone was located in the central region and had to be passed throughbriefly in going from 95° C. to 60° C. Detection was made post-PCR usinggel electrophoresis. Since this PCR technique is not based on heatingthe entire surfaces of the reaction vessel, the reaction rate isdetermined by a flow rate, not a heating/cooling rate.

Park et al. (Anal Chem 75:6029-6033 (2003)) describe a continuous-flowPCR device that uses a polyimide coated fused silica capillary wrappedinto a helix around three temperature-controlled blocks. Sample volumeswere 2 μl. Detection was made post PCR using gel electrophoresis.Reference was made to the possibility of adapting their instrument forreal-time PCR by using a capillary coated with PTFE instead ofnon-transparent polyimide. See also, Hahn et al. (WO 2005/075683).

Enzelberger et al. (U.S. Pat. No. 6,960,437) describe a microfluidicdevice that includes a rotary channel having three temperature zones. Anumber of integrated valves and pumps are used to introduce the sampleand to pump it through the zones in a rotary fashion.

Knapp et al. (U.S. Patent Application Publication No. 2005/0042639)describe a microfluidic device capable of single molecule amplification.A planar glass device with several straight parallel channels isdisclosed. A mixture of target DNA and PCR reagents is injected intothese channels. In a first embodiment, the channels are filled with thismixture and flow is stopped. Then the entire length of the channels isthermally cycled. After thermal cycling is completed, the channels areimaged in order to detect regions of fluorescence where DNA has beenamplified. In a second embodiment, the PCR mixture flows continuouslythrough the amplification zone as the temperature is cycled, andfluorescence is detected downstream of the amplification zone. Differentdegrees of amplification are achieved by altering the time spent incycling, through changing distance traveled under cycling, and the like.It is worth noting that this method varies conditions (such as cyclesexperienced) for separate consecutive sample elements, rather thanmonitoring the progress of individual sample elements over time.

US 2005/0129582A1, entitled System and method for heating, cooling andheat cycling on microfluidic device, describes an integrated heatexchange system on a microfluidic card. According to one aspect of thesystem, the portable microfluidic card has a heating, cooling, and heatcycling system on-board such that the card can be used portably. Themicrofluidic card includes one or more reservoirs containing exothermicor endothermic material.

Kohler et al. (U.S. Pat. No. 6,896,855) discusses a miniaturizedtemperature zone flow reactor that has fixed (non adjustable)temperature zones that can be used for thermocycling.

Various additional attempts have been made to develop an adequate devicefor monitoring and changing the temperature on a microfluidic device.For example, International Patent Application PCT/US98/1791 discussesdevices that controls and monitors temperature within microfluidicsystems by applying electric currents to fluids to generate heattherein, as well as measure solution conductivity as a measure of fluidtemperature.

Another system for controlling temperature on a microfluidic device isdescribed in U.S. Pat. No. 6,541,274. This patent is directed to areactor system having a plurality of reservoirs in a substrate. A heatexchanger is inserted in the reservoirs to control the temperature.Still other examples of existing devices for controlling temperature ona microfluidic device is with radiant heat as described in U.S. Pat. No.6,018,616, and the temperature regulated controlled block as describedin U.S. Pat. No. 6,020,187.

SUMMARY

The present invention provides systems and methods for performing athermocyclic process, such as amplifying nucleic acids.

Aspects of the invention are embodied in an apparatus for performing athermocyclic process comprising a microfluidic chip having a fluidchannel formed therein and one or more thermal distribution elements.The microfluidic chip is configured with two or more temperature zones.Each thermal distribution element is in thermal communication with anassociated portion of the microfluidic chip, and each thermaldistribution element is constructed and arranged to distribute thermalenergy from an external thermal energy source substantially uniformlyover the associated portion of the microfluidic chip, thereby definingthe associated portion as one of the temperature zones within saidmicrofluidic chip. The channel is arranged such that a fluid flowingthrough the channel would enter and exit each of the temperature zonesof the microfluidic chip a plurality of times.

Still other aspects of the invention are embodied in a system forperforming a thermocyclic process. The system comprises a microfluidicchip having a fluid channel formed therein, a thermal energy source, anddetectors. The microfluidic chip is configured with two or moretemperature zones and includes one or more thermal distribution elementsin thermal communication with an associated portion of the microfluidicchip. Each thermal distribution element is constructed and arranged todistribute thermal energy from an external thermal energy sourcesubstantially uniformly over the associated portion of the microfluidicchip, thereby defining the associated portion as one of the temperaturezones within the microfluidic chip. The channel is arranged such that afluid flowing through the channel would enter and exit each of thetemperature zones of the microfluidic chip a plurality of times.

A thermal energy source is associated with each thermal distributionelement and is configured to apply thermal energy to the associatedthermal distribution element. The detector is configured to detectemissions originating from one or more locations within the channel.

Still other aspects of the invention are embodied in a DNA amplificationmethod. The method comprises providing an apparatus for amplifying DNA.The apparatus comprises a microfluidic chip having a channel, a firstthermal distribution element, and a second thermal distribution element.The first thermal distribution element covers only a first portion ofthe microfluidic chip, and the second thermal distribution elementcovering only a second portion of the microfluidic chip that does notoverlap with the first portion of the microfluidic chip. The first andsecond thermal distribution elements are arranged such that there is agap between the first and second thermal distribution elements, the gapcorresponding to a third portion of the microfluidic chip. The channelis configured such that a fluid flowing through the channel would enterand exit the first, second, and third portions of the microfluidic chipa plurality of times.

Thermal energy is applied to the first thermal distribution element togenerate a first temperature in the first portion of the microfluidicchip, and thermal energy is applied to the second thermal distributionelement to generate a second temperature in the second portion of themicrofluidic chip. A third temperature may be generated in the thirdportion of the microfluidic chip. A solution containing a nucleic acidsample is pumped through the channel so that the solution alternatelyflows through the first, second, and third portions of the microfluidicchip and is alternately exposed to the first, second, and thirdtemperatures.

The method further includes, while the solution is being pumped,detecting emissions originating from solution flowing through a portionof the channel disposed within the one of the portions of themicrofluidic chip.

The above and other aspects and embodiments of the present invention aredescribed below with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIG. 1 is a block diagram illustrating a system according to someembodiments of the invention.

FIG. 2 is a perspective view of an exemplary arrangement of heaters anda microfluidic chip with thermal distribution elements embodying aspectsof the present invention.

FIG. 3 is a side view of the arrangement shown in FIG. 2.

FIG. 4 is a perspective view of the microfluidic chip of FIG. 2 andillustrates three temperature zones that are defined by the arrangementshown in FIGS. 2 and 3.

FIG. 5 is an exemplary temperature profile showing temperature cyclingthat can be achieved in accordance with aspects of the presentinvention.

FIGS. 6A-6D are plan views of microfluidic chips illustrating otherexemplary arrangements of thermal distribution elements.

FIG. 7 is a bottom perspective view of a microfluidic chip showing amachine-readable bar code on the bottom of the chip.

FIG. 8 is a perspective view of heaters and a microfluidic chip withthermal distribution elements illustrating that at least a portion of adefined temperature zone may be within the field of view of a detector.

FIG. 9A is a perspective view of an alternative arrangement of heatersand a microfluidic chip with thermal distribution elements embodyingaspects of the present invention.

FIG. 9B is a side view of the arrangement shown in FIG. 9A.

FIGS. 10A-10B illustrate various possible arrangements of a detector incombination with the arrangement of FIGS. 9A and 9B.

FIG. 11 illustrates an exemplary temperature profile.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, the words “a” and “an” mean “one or more.” Furthermore,unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice of the present invention, the preferred materials andmethods are described herein.

FIG. 1 illustrates a block diagram of a system 100 for performing athermocyclic process, such as real-time PCR, with which the presentinvention may be implemented. The system 100 may include a source ofsample material, such as test solution reservoir 101, which may containmultiple test solutions, e.g., test samples. The system may furtherinclude a source of carrier fluid, such as carrier fluid reservoir 103.The solution fluid reservoir 101 and/or the carrier fluid reservoir 103may comprise chambers within a cartridge that is coupled to themicrofluidic device, such as the cartridge described incommonly-assigned U.S. patent application Ser. No. 11/850,229, thedisclosure of which is hereby incorporated by reference in its entirety.

The test solution may be substantially the same as the carrier fluid,except that the test solution comprises all the necessary real-time PCRreagents. The real-time PCR reagent mixture may include, for example,PCR primers, dNTPs, polymerase enzymes, salts, buffers,surface-passivating agents, and the like. In addition, the real-time PCRmixture may include a non-specific fluorescent DNA detecting molecule, asequence-specific fluorescent DNA probe, or a marker. The carrier fluidmay be an immiscible fluid (such as an oil, a fluorinated liquid, or anyother nonaqueous or hydrophobic solvent). The purpose of the carrierfluid is to deter transfer of material from one test bolus to another.Another purpose of the carrier fluid is to provide a distinguishabletransition between boluses that may be used to track the fluid flow inthe channel. The carrier fluid may include a marker.

The test solution and carrier fluid are introduced into a microchannel202 of a microfluidic chip 200, for example, through a switch 104. Thedimensions of the microchannel are small enough to allow for theamplification and detection of a single DNA molecule originally presentin the test solution. Switch 104 may be under the control of a maincontrol and processing computer 105 such that the carrier fluid and thetest solution are sequentially, alternately introduced into microchannel202. The volume of the test solution and carrier fluid that isintroduced into microchannel 202 is selected such that there is minimalblending between them during movement through microchannel 202.

Alternatively, sample material may be provided as a continuous stream inthe microchannel 202, and assay-specific reagents and buffer materialmay be alternately introduced into the continuous stream of samplematerial, e.g., to create a sequential arrangement of discrete testboluses flowing through the microchannel.

A multitude of reactions in series (or sequential reactions) can thus becarried out in the same microchannel 202 as a result of the continuousmovement of different test solutions through microchannel 202, eachseparated by the carrier fluid. The flow rate of the carrier fluid andtest solution through microchannel 202 is controlled by pump mechanism106. Pump mechanism 106 is under the control of main control andprocessing computer 105 in order to regulate the flow rate of the testsolution and the carrier fluid in microchannel 202.

Pump mechanism 106 can regulate the flow rate of the test solution andcarrier fluid by positive pressure at the upstream side or inlet ofmicrochannel 202 or by negative pressure at the downstream side oroutlet of microchannel 202. In one embodiment, the pressure differenceis approximately 1 psi, although other pressure differences may beutilized.

A temperature control system 107 is included in the system to controlthe temperature to produce suitable temperatures for the PCR cycles asthe test solution moves through microchannel 202. Suitable temperaturesfor the PCR cycles are well known to skilled artisan and may include afirst temperature in the range of about 85° C. to about 100° C., asecond temperature in the range of about 20° C. to about 70° C., and athird temperature in the range of about 55° C. to about 80° C.Temperature control system 107 may include thermal elements (i.e.,thermal energy sources), such as heaters 108 and/or coolers 109,temperature sensors 110, and a temperature controller 111. Temperaturecontroller 111 collects temperature information from the temperaturesensors 110, and generates control signals based on the temperatureinformation. Temperature controller 111 is under control of main controland processing computer 105 so that the desired temperatures aremaintained in the heaters 108 and/or coolers 109.

Heating and cooling may be accomplished by circulating water or fluidbaths or by Peltier-effect elements which are well known to the skilledartisan.

System 100 may further include an optical imaging system 112, whichdetects emissions (e.g., fluorescence or chemiluminescence) which areindicative of the presence—and possibly the amount—of a nucleic acid ofinterest (i.e., amplification products) and to monitor the flow rate ofthe test solution in microchannel 202. In one embodiment, the opticalimaging system 112 is a fluorescent imaging system that preferablyincludes one or more excitation sources 113, one or more optics/filtersmodules 114, and one or more detectors 115. The excitation sources 113generate light at desired wavelengths to excite the labels used fordetecting the amplification products during real-time PCR and/or todetect markers that may be present to monitor the flow rate of the testsolution in microchannel 202. In addition to filters, optics/filters 114may include, lenses, light pipes, mirrors, beam splitters, etc. and areused to form a beam of light and/or to direct the light from excitationsources 113 to the appropriate positions on the microchannel 202.Optics/filters 114 are also used to direct a portion of the emissionlight toward the detectors 115 and to filter the light to exclude lightof undesired wavelengths or to reduce backscatter from reachingdetectors 115. The desired wavelengths to excite the labels used inreal-time PCR will depend on the precise labels and/or markers used,e.g., intercalating dyes, molecular beacons, quantum dots or TaqMan®probes, which wavelengths are well known to skilled artisans. Similarly,the emission wavelengths of the precise labels and/or markers are wellknown to skilled artisans. Detectors 115 detect the emission wavelengthsof the excited labels and/or markers and measure the intensity of theemitted light. Optical imaging system 112 preferably is able todistinguish between multiple microchannels in a microfluidic device.

Optical imaging system 112 is under control of main control andprocessing computer 105 which directs the optical imaging system 112 tomeasure the intensity of the emitted light at desired time intervals,such as, for example, at least once during each PCR cycle at one or aplurality of locations in microchannel 202. Detectors 115 generate asignal or an image of the intensity of the emitted light and direct itto main control and processing computer 105 for analysis of theamplification product and for monitoring the flow rate of the testsolution. Detectors 115 may include multiple-pixel array detectors (suchas a CMOS or CCD detector) and/or discrete single-pixel or non-imagingdetectors. Detectors 115 may be integral with or proximal tomicrochannel 202 or to the microchannels of a microfluidic device.Detectors 115 may be stationary or may be scanning. The detectors 115should have appropriate resolution for obtaining meaningful results andfor monitoring of fluid flow in microchannel 202, particularly becausethe fluid is continuously moving in microchannel 202.

The real-time PCR mixture may include a non-specific fluorescent DNAdetecting molecule (such as an intercalating dye), a sequence-specificfluorescent DNA probe (such as a molecular beacon, a TaqMan® probe, or aquantum dot probe), or a flow marker (such as a quantum dot), and thecarrier fluid may include a flow marker. In one embodiment, the opticalimaging system 112 is utilized to detect the intensity of thefluorescence from the DNA detecting molecule or the probe (i.e., theintensity of the fluorescent signal) and/or to detect the fluorescenceof the marker. The fluorescence of the marker can be used to delineatethe test solution from the carrier fluid and can also be used todetermine and monitor the flow speed of the test solution or carrierfluid. The intensity of the fluorescent signal can be used to detectamplified product, to determine the quantity of amplified product, todetermine the number of original molecules present in the test solution,and the like as well known to a skilled artisan for real-time PCR. Theintensity of the fluorescent signal can also be used to determine andmonitor the flow speed of the test solution.

The intensity of the fluorescent signal may be measured (e.g., an imageof the fluorescent signal is taken) at a specific time and/ortemperature during the PCR temperature cycle. Alternatively, theintensity of the fluorescent signal can be measured once during each PCRcycle.

After test solution has moved through microchannel 202 and completed thedesired number of PCR cycles, it may optionally be sent to a post-PCRanalyzer 116. Post-PCR analyzer 116 may include any analytical techniquethat can be used on PCR amplification products. Such techniques include,but are not limited to, sequencing, electrophoresis, probing, thermalmelt curve analysis, and the like.

Aspects of the present invention are embodied in an arrangementincluding thermal elements 208 and 210 in combination with amicrofluidic chip 200 having thermal distribution elements 204, 206 asshown in FIG. 2. The microfluidic chip 200 may be fabricated in anysuitable size, preferably in the range of 1 cm² to 100 cm², and mostpreferably approximately 20 mm×20 mm, and is ideally manufactured fromany suitable material, including borosilicate glass, quartz glass,plastic polymers, and silicon. One or more microfluidic channels, ormicrochannels, 202 are formed on the surface of the chip 200 for movingsample solution therethrough. The microfluidic channel 202 is preferablyarranged in a serpentine manner extending transversely across thesurface of the chip 200 substantially from one side of the chip to theother and back.

The microfluidic channel 200 traverses through the chip from an inletside to an outlet side and is moved via typical and known means ofpressure-driven flow. Flow rates may vary between about 10 nanolitersper minute to about 1 milliliter per minute. The serpentine pattern ofthe microchannel is repeated numerous times to provide sample exposureto PCR temperature cycles. In one embodiment, the number of across andback serpentine patterns is approximately 40, but can be in the range of20 to 60. A preferred embodiment includes 30 to 50 serpentine patternsand most preferably includes 30 to 40 serpentine patterns.

Thermal distribution elements 204 and 206 are secured to the chip 200overlying portions of the microfluidic channel 202. In the embodimentshown in FIG. 2, the thermal distribution elements 204 and 206 areelongated plates formed from a thermally conductive material, preferablya metal, most preferably copper (less preferably aluminum), and arepositioned on the chip 200 so as to extend substantially from one end ofthe chip to the opposite end of the chip. The thermal distributionelements 204 and 206 are preferably secured to the microfluidic chip 200by a thermally conductive adhesive, such as a thermally conductiveepoxy.

The heating elements 208 and 210 may comprise elements of the heaters108 and/or coolers 109 of the temperature control system 107 (see FIG.1). The heaters, or thermal generating devices, are capable of applyingheat or cold to the microfluidic chip. Heaters 208 and 210 may compriseany type of heating device including resistive, thermal electric,Peltier, etc. The temperatures of the heaters 208 and 210 are preciselycontrolled at fixed temperatures and have a thermal mass greater thanthat of the chip 200. Temperature control is maintained by thetemperature sensors 110 of the temperature control system 107 (see FIG.1), which may include any suitable type of temperature measuringdevices, such as thin film resistors, thermistors, or thermocouples.

As shown in FIGS. 2 and 3, heaters 208 and 210 are placed in contactwith the thermal distribution elements 204 and 206, for example, byplacing the microfluidic chip 200 into the system 100 shown in FIG. 1.Thermal energy generated by the heaters 208 and 210 is transferred, viathe thermal distribution elements 204 and 206, respectively, to themicrofluidic chip 200. The thermal distribution elements 204 and 206function to not only transfer thermal energy from the heaters 208 and210 to the microfluidic chip 200, but also to provide a substantiallyuniform distribution of the transferred thermal energy across a discretearea defined by the portion of the thermal distribution element incontact with the chip. The microfluidic chip 200 is preferably made froma material that is a poor thermal conductor, and thus, the thermalenergy transferred to the microfluidic chip 200 via the thermaldistribution elements 204 and 206 is substantially confined to the areaof the thermal distribution element that is in contact with themicrofluidic chip 200.

Accordingly, it can be appreciated that the arrangement illustrated inFIGS. 2 and 3 creates three distinct temperature zones. This isillustrated in FIG. 4. A first zone (“zone 1”) corresponds to thatportion of the microfluidic chip 200 covered by the thermal distributionelement 206. A second zone (“zone 2”) corresponds to that portion of themicrofluidic chip 200 that is covered by the thermal distributionelement 204. Finally, a third zone (“zone 3”) is defined by that portionof the microfluidic chip 200 that is between the portions covered bythermal distribution elements 204 and 206 and is not in contact with athermal distribution element. It can be further appreciated that asmaterial flows through the serpentine path of the microchannel 202, thefluid will sequentially pass through zones 1, 3 and 2 a number of timesas the fluid traverses the length of the microchannel 202.

The temperature of the third zone may be controlled by other meansincluding flowing air of a controlled temperature passing over themicrofluidic chip 200.

To effect PCR on sample fluid flowing through the microfluidic channel202, heater device 210 would typically be adjusted to approximately 94°C. to achieve the denaturation temperature of the PCR in zone 1. Heaterdevice 208 would typically be adjusted to approximately 50° C. toachieve the annealing temperature of the PCR in zone 3. Accordingly,fluid sample flowing through the microchannel 202 will pass through thezones 1, 2 and 3, and thus will be exposed to the discrete temperaturesof each of those zones thereby effecting rapid temperature transitionsto achieve the necessary thermal cycling to accomplish PCR.

FIG. 5 shows a typical temperature profile showing the temperaturetransition that can be accomplished with an arrangement such as thatshown in FIGS. 2, 3 and 4. In the illustrated profile, the samplematerial is exposed to the temperature of zone 1 (T1) for a period oftime required to traverse across and back zone 1. From zone 1, thesample flows into zone 3 and is exposed to the temperature of zone 3(T3) for a period of time required to cross zone 3. From zone 3, thesample flows into zone 2 and is exposed to the temperature of zone 2(T2) for a period of time required to traverse across and back zone 2.From zone 2, the sample flows back into zone 3 where it is exposed totemperature T3. The period of exposure at each temperature will dependon the length of microchannel within the corresponding temperature zoneand the sample flow rate.

Examples of thermal distribution element location and size variationsare shown in FIGS. 6A through 6D. As sample materials move linearlythrough the serpentine microchannels, the sample traverses the changingtemperature-controlled regions. The length of time within a giventemperature zone, dwell time, is controlled by the width (top to bottomin FIGS. 6A through 6D) of the thermal distribution element definingthat zone.

FIG. 6A shows a microfluidic chip 220 having a serpentine microchannel222 and relatively small thermal distribution elements 224 and 226 thatare approximately of the same size. Accordingly, the temperature regionsdefined by thermal distribution elements 224 and 226 are relativelysmall as compared to the temperature region defined between the thermaldistribution elements. Thus, the dwell times at the temperaturescorresponding to the zones defined by thermal distribution elements 224and 226 is less than that in the temperature zone defined between thethermal distribution elements.

In FIG. 6B, microfluidic chip 230 includes a serpentine microchannel 232and thermal distribution elements 234 and 236 which are approximatelythe same size and are relatively wide as compared to the temperatureregion defined between the thermal distribution elements. Accordingly,the dwell times in the temperature zones defined by thermal distributionelements 234 and 236 would be greater than the dwell time within thetemperature zone defined between the thermal distribution elements.

In FIG. 6C, microfluidic chip 240 includes a serpentine microchannel 242and thermal distribution elements 244 and 246. Thermal distributionelement 246 is larger than thermal distribution element 244.Accordingly, dwell time within the temperature zone defined by thethermal distribution element 246 would be greater than the dwell timewithin the temperature zone defined by thermal distribution element 244.

In FIG. 6D, microfluidic chip 250 includes a serpentine microchannel 252and thermal distribution elements 254 and 256. Thermal distributionelement 254 is larger than the thermal distribution element 256.Accordingly, the dwell time within the temperature zone defined bythermal distribution element 254 would be greater than a dwell timewithin the temperature zone defined by thermal distribution element 256.

As can be appreciated by persons of ordinary skill in the art, manydwell time profiles can be derived by changing the size and shape of thethermal distribution elements. By adjusting the width of the thermaldistribution element (where the microchannel is oriented so that sampleflowing through the microchannel traverses across the width of thethermal distribution element), the size of the temperature zone isproportionally adjusted. The effect is a change in the dwell time of thePCR cycle due to the fact that the sample drop will move through themicrochannel at a substantially constant velocity. This would permitadjustment to a particular, optimized assay. And thus, different assaychips could then be fabricated with different dwell time profiles bycontrolling the size and shape of the thermal distribution elements aswell as the orientation of the microchannel(s) relative to the thermaldistribution element.

An advantage of the instant invention is that microfluidic chips can beprepackaged for optimized assay designs, e.g. specificdiagnostic/analytical tests, that optimize the PCR reaction. As shown inFIG. 7, during chip fabrication, a label 300 can be affixed to the chipso that the instrument can readily identify the chip via an opticaldetector reading the label. Preferably, the labeling is machinereadable, for example, a bar code or RFID tag. The instrument will thenadjust the assay and instrument process control to that of the specificPCR protocol mandated by the inserted chip. The microfluidic chips arepreferably disposable.

The arrangement described herein utilizes a purposeful open centersection, previously described as zone 3. This section preferably definesthe temperature region wherein the PCR amplification extension phaseoccurs. The section is optically clear to allow fluorescence excitationand detection apparatus to probe and measure the microchannel areacontinuously without interference from the heating mechanisms or thethermal distribution elements. Zone 3 is typicallytemperature-controlled to approximately 74° C., for example, by flowingair at the same temperature over zone 3.

Other embodiments of the invention permit zone 3 to consist ofalternating temperatures to attain a true three-step PCR reaction. Instill further embodiments, a two-step PCR reaction is possible byminimizing the size of zone 3 such that the sample passes immediatelyfrom zone 1 to zone 2.

Real-time PCR measurements are typically performed at the end of theextension phase of the PCR cycle. The microfluidic chip described hereinfacilitates this by design. As shown in FIG. 8, the optical imagingsystem 112 can be positioned and configured so as to be directed at zone3, which is unobstructed by heating elements 208, 210 or thermaldistribution elements 204, 206.

FIGS. 9A and 9B illustrate an arrangement whereby zone 3 (i.e., the zonewithout a thermal distribution element) is positioned at a locationother than the center of the chip, for example along one edge of thechip as shown. In the arrangement shown, microfluidic chip 260 includesa microchannel 262 and thermal distribution elements 264 and 266.Thermal distribution element 264 is a generally plate-like paneldisposed over portion of the chip so as to cover a portion of themicrochannel 262. Thermal distribution element 266 is in the form of anelongated rectangular block having a length substantially the same asthat of thermal distribution element 264, but a thickness substantiallygreater than that of thermal distribution element 264. Heating element268 is placed in contact with thermal distribution element 264.Furthermore, heating element 270 is placed in contact with thermaldistribution element 266, and because thermal distribution element 266is thicker than heating element 268, heating element 270 can be placedabove heating element 268 without contacting heating element 268.

FIGS. 10A and 10B show the arrangement illustrated in FIGS. 9A and 9Band identify the temperature zones defined by the thermal distributionelements 264 and 266 and the portion of the microfluidic chip 260 thatis uncovered by a thermal distribution element. FIGS. 10A and 10B alsoillustrate possible positions for an optical detection system withrespect to the microfluidic chip. As shown, the optic devices formeasuring real time PCR fluorescence can be physically located above orbelow the chip. Furthermore, as shown in FIG. 10B, the location of theoptic device below the chip for the purpose of measuring thefluorescence can be at any PCR zone location, for example zone 2, asshown, which is below thermal distribution element 266 and heater 270.

A further embodiment of the invention details a four-step PCR process.If the area of zone 3 of the arrangement shown in FIGS. 2 and 3 issimply adjusted to the extension temperature, e.g., approximately 74°C., then a four-step PCR process would be generated. A temperatureprofile for such an arrangement is shown in FIG. 11.

While various embodiments/variations of the present invention have beendescribed above, it should be understood that they have been presentedby way of example only, and not limitation. Thus, the breadth and scopeof the present invention should not be limited by any of theabove-described exemplary embodiments. Further, unless stated, none ofthe above embodiments are mutually exclusive. Thus, the presentinvention may include any combinations and/or integrations of thefeatures of the various embodiments.

Additionally, while the processes described above and illustrated in thedrawings are shown as a sequence of steps, this was done solely for thesake of illustration. Accordingly, it is contemplated that some stepsmay be added, some steps may be omitted, and the order of the steps maybe re-arranged.

What is claimed is:
 1. A DNA amplification method, comprising: providingan apparatus for amplifying DNA, the apparatus comprising: amicrofluidic chip having a channel; a first thermal distribution elementcovering only a first portion of the microfluidic chip; and a secondthermal distribution element covering only a second portion of themicrofluidic chip that does not over lap with the first portion of themicrofluidic chip, wherein the first and second thermal distributionelements are arranged such that there is a gap between the first andsecond thermal distribution elements, the gap corresponding to a thirdportion of the microfluidic chip, and wherein the channel is configuredsuch that a fluid flowing through the channel would enter and exit thefirst, second and third portions of the microfluidic chip a plurality oftimes; applying thermal energy to the first thermal distribution elementto generate a first temperature in the first portion of the microfluidicchip; applying thermal energy to the second thermal distribution elementto generate a second temperature in the second portion of themicrofluidic chip; pumping a solution containing a nucleic acid samplethrough the channel so that the solution alternately flows through thefirst, second, and third portions of the microfluidic chip and isalternately exposed to at least the first and second temperatures. whilethe solution is being pumped, detecting emission originating fromsolution flowing through a portion of the channel disposed within one ofthe first, second, and third portions of the microfluidic chip.
 2. Themethod of claim 1, further comprising maintaining a third temperaturewithin the third portion of the microfluidic chip and determining adesired width of the third portion of the microfluidic chip, wherein thewidth is determined based, at least in part, on a desired dwell time atthe third temperature.
 3. The method of claim 1, further comprising:determining a desired first width of the first portion of themicrofluidic chip, wherein the first width is determined based, at leastin part, on a desired dwell time at the first temperature; applying thefirst thermal distribution element having the first width to the firstportion of the microfluidic chip; determining a desired second width ofthe second portion of the microfluidic chip, wherein the second width isdetermined based, at least in part, on a desired dwell time at thesecond temperature; and applying the second thermal distribution elementhaving the second width to the second portion of the microfluidic chip.4. The method of claim 1, wherein applying thermal energy to the firstthermal distribution element comprises connecting a first heater to thefirst thermal distribution element, and applying thermal energy to thesecond thermal distribution element comprises connecting a second heaterto the second thermal distribution element.
 5. The method of claim 4,wherein the first thermal distribution element comprises a thermallyconductive metal plate that is secured to the first portion of themicrofluidic chip, the second thermal distribution element comprises athermally conductive metal plate that is secured to the second portionof the microfluidic chip, the thermal mass of the first heater isgreater than the thermal mass of the microfluidic chip, and the thermalmass of the second heater is greater than the thermal mass of themicrofluidic chip.
 6. The method of claim 1, wherein the channel isconfigured such that a fluid flowing through the channel would enter andexit the first, second and third portions of the microfluidic chip atleast 10 times.
 7. The method of claim 3, wherein the combined widths ofthe first thermal distribution element and the second thermaldistribution element substantially equals the width of the portion ofthe microfluidic chip covered with the microchannel, such that the thirdportion is so small and fluid flowing through the microchannel flowsthrough the third portion so quickly that the fluid is exposed tosubstantially only the first and second temperatures for dwell timesdetermined by the first and second thermal distribution elements,respectively.